Recombinant Expression and Refolding of a Potassium Channel-Activating Three-Finger Toxin

Abstract

Tx7335 is a novel three-finger peptide toxin that was originally isolated from the Eastern Green Mamba venom. A unique characteristic of Tx7335 is the ability to increase duration and frequency of openings in the potassium channel KcsA. Potassium channels play an important role in electrical signaling of excitable cells. Dysfunction of the channels can lead to major health problems such as certain heart and autoimmune diseases. Potassium channels are natural targets for venomous animals seeking to paralyze their prey. As a result, toxins have been shown to be an effective method of modulating and studying potassium channels. To obtain sufficient amounts of Tx7335 for future studies into its mechanism of action, we are recombinantly expressing and purifying this toxin. A synthetic gene encoding for Tx7335 was cloned into an expression vector expressing the toxin as a thioredoxin fusion protein with a TEV protease cleavage site in between them. The fusion protein was overexpressed in E coli. BL21(DE3) C43 expression strain and purified by Nickel affinity chromatography. The toxin was released from the fusion protein by cleavage with TEV protease and refolded by addition of reduced and oxidized glutathione in 1 to 10 ratio. Tx7335 was further purified through Cation exchange chromatography. Refolding was assessed by mass spectrometry, and the recombinant toxin will be used for future functional and structural studies of toxin-KcsA interactions.